Appropriate differentiation and development of higher organisms require precisely regulated expression of multiple genes. The primary control for most genes is exerted at the level of transcription. This involves the combinatorial action of tissue-specific and ubiquitous transcription factors acting at regulatory sequences that are proximal (promoters) or distal (enhancers, insulators, silencers, and locus control regions [LCRs]) to a gene. The existence of functionally distinct cis-acting elements indicates that the high degree of regulation involved in coordinated gene expression within a complex organism requires more intricate circuitry than a simple promoter can provide to turn genes on and off. A critical aspect of this circuitry and coordination is the regulation imposed upon genes within a complex nuclear environment.
The human genome, however, is composed of about 3.3×109 bp. If stretched out, this would represent a length of more than 1.8 meters of DNA. The cell nucleus that contains two copies of this DNA is, on the other hand, a sphere of no more than 6 μm in diameter. To reach this high level of compaction, human DNA is, like in all other eukaryotes, organized into chromatin. The packaging of DNA into chromatin within the eukaryotic nucleus is highly organized and plays a critical role in regulating gene expression and other nuclear processes. The basic structural unit of chromatin is the nucleosome, which consists of ˜146 bp of DNA wrapped in 1.75 superhelical turns around a histone octamer containing two molecules each of histones H2A, H2B, H3 and H4. This unit is repeated once every 200+/−40 bp as a nucleosomal array in chromosomal DNA. The array is further compacted into a higher-order structure by the association of histone H1 with nucleosomes within the array.
The functional consequence of chromatin packaging, in general, is to restrict access of the DNA to a variety of DNA-binding proteins that regulate gene activity. Biochemical and genetic evidence amply demonstrate that nucleosomes are normally repressive for transcription. Several elegant mechanisms have evolved, however, that modulate chromatin structure to increase the accessibility of DNA for protein interaction. These pathways involve distinct protein complexes that function either as motors to disrupt nucleosomes (ATP-driven chromatin remodeling complexes) or as enzymatic machinery to chemically modify histones (histone acetyltransferases and deacetylases). Such mechanisms may be critical in programming genes to be either active or inactive in a particular cell type or to be poised for expression at a specific stage of development or in response to environmental signals.
Chromatin structural changes can occur at several levels: either globally by the decondensation (active nucleic acids) or condensation (inactive nucleic acids) of a large chromosomal domain or locally by the disruption (active) or formation (inactive) of one or more nucleosomes on a promoter or enhancer region. Global chromatin structural changes have been shown to occur in the human β-globin gene locus by the action of the distal LCR (Forrester, W. C. et al., Genes Dev., 4, 1637 (1990)). In addition, active genes are characterized by containing hyperacetylated histones and undermethylated DNA. Interestingly, both global and local levels of chromatin structural perturbation often require the interaction of regulatory proteins with histone amino-terminal tails within the nucleosome, which are also the main targets of post-translational modification (for review, see Davie, J. R., Curr. Opin. Nucleic Genet. Dev., 8, 173 (1998)). Two critical pathways that facilitate this interaction involve distinct protein complexes that function either as motors to disrupt nucleosomes (ATP-driven chromatin remodeling complexes) or as enzymatic machinery to modify histones chemically and alter their affinity for DNA (histone acetylases, HATs/deacetylases, HDACs/kinases).
The mechanisms by which specific genes are activated in chromatin have been extensively investigated in a variety of biochemical and genetic systems. Paranjape, S. M. et al., Annu. Rev. Biochem., 63, 265 (1994). Recent in vitro studies have focused on the role of specific cellular and viral factors in chromatin structural reconfiguration and nucleic acid expression. A common theme to emerge is that chromatin remodeling and transcriptional activation are separate processes that can be regulated by distinct proteins or subunits/domains of a given protein. This was shown originally with the GAL4-VP16 activator using chromatin-assembled genes. Pazin, M. J. et al., Science, 266, 2007 (1994). The observation that chromatin accessibility is not sufficient for transcription has important regulatory implications as nucleic acids can be preset by chromatin remodeling to be activated at a later time.
There are seven chromatin remodeling complexes that have been described to date: SWI/SNF, RSC, NURF, CHRAC, ACF, NURD and RSF. All are multi-subunit complexes with molecular weights ranging from 2 MDa to 0.5 MDa. Biochemical analyses have shown that these complexes can disrupt nucleosomal structure in a ATP-dependent manner (all complexes), facilitate factor binding (SWI/SNF, NURF, ACF) and transcription from chromatin-assembled genes (NURF, ACF and RSF). Several properties indicate that these complexes are functionally and mechanistically distinct. For example, RSC is an abundant complex in yeast and is encoded by essential genes, in marked contrast to SWI/SNF (SWI stands for mating type SWItch and SNF for Succrose Non-Fermenting), suggesting a different biological role for these two complexes. Cairns, B. R. et al., Cell, 87, 1249 (1996). Furthermore, NURF has recently been shown to facilitate transcriptional activation from preformed chromatin templates in combination with GAL4-HSF. Mizuguchi, G. et al., Mol. Cell. 1, 141 (1998). In this assay, NURF cannot be replaced by either yeast SWI/SNF or CHRAC.
A novel complex has recently been described, using a purified in vitro transcription system, that alleviates the nucleosomal block to elongation. Orphanides, G. et al., Cell, 92, 105 (1998). This 230 kDa complex, called FACT (facilitates chromatin transcription), appears to function quite distinctly from chromatin remodeling complexes as it does not facilitate transcriptional initiation or require ATP hydrolysis. Thus, promoter-proximal chromatin remodeling is one critical step in gene activation but is not sufficient for transcription unless coupled with activities, such as FACT, which permit efficient elongation through nucleosomes.
Little is known about the manner in which remodeling complexes disrupt nucleosomes. Recent studies demonstrate that the ability of NURF to alter nucleosomal structure is impaired by crosslinking of nucleosomal histones, removal of amino-terminal histone tails, or mutation of lysine residues within the histone H4 tail; this indicates that the flexible tails play a critical role in the remodeling process. Georgel, P. T. et al., EMBO. J., 16, 4717 (1997). The formation of a ternary complex composed of DNA, histones, and activator is facilitated by SWI/SNF, which results in the destabilization but not the loss of nucleosomes and persists after its removal. Owen-Hughes, T. et al., Science, 273, 513 (1996). Recent studies demonstrate that the SWI/SNF-dependence of genes regulated by the yeast activator GAL4 is determined by the presence of low, rather than high, affinity GAL4 DNA binding sites on the promoter. The presence of high-affinity sites or a nucleosome-free region can overcome the requirement for this remodeling complex in vivo. Burns, L. G. et al., Mol. Cell. Biol., 17, 4811 (1997).
Interestingly, one subunit of NURF has recently been identified as the WD repeat protein, p55, which is also a subunit of Drosophila CAF1 (chromatin assembly factor 1). p55 homologs are found associated with histone acetyltransferases and deacetylases. Thus, many of the diverse chromatin-altering complexes may utilize common subunits. Martinez-Balbas, M. A. et al., Proc. Natl. Acad. Sci. USA, 95, 132 (1998).
It is clear that multiple levels of control are involved in regulated nucleic acid expression, from the activation of the chromosomal domain in which a nucleic acid resides to the formation of a basal initiation complex on a given promoter within the domain. Questions remain as to how tissue- or developmental-state-specific expression is established and how coordinate expression of multiple genes is achieved. In addition, the mechanism by which critical DNA control elements, often acting at long-range, such as enhancers, insulators, silencers, and LCRs, regulate transcription is still poorly understood.
Thus, there remains a continuing need for high-throughput screening assays that identify small molecule compounds that enhance or block the association between chromatin remodeling complexes and the specific transcription factors with which they interact, such as the BRG1 subunits of the SWI/SNF complex and proteins containing zinc finger motifs. In this way very specific drugs are developed that modulate the activation or repression of selective nucleic acids that are regulated by SWI/SNF (BRG1) complexes and zinc finger DNA-binding transcription factors. There also remains a need for a method of treating nucleic genetic diseases where nucleic acid expression is targeted in a highly selective manner.